The ischemic penumbra was originally defined as peri-infarct tissue with partial reduced blood flow. The blood flow reduction (30-60% of control values) was less severe than in the ischemic core allowing the penumbral tissue to maintain normal transmembrane ionic gradients. The electrical silence of the penumbra was ascribed to the partial reduction in substrate delivery, e.g. the energy supply was sufficient to maintain membrane potential, but not to support synaptic transmission. The fate of astrocytes in the penumbra has received little attention, in part, because the activity of these non-excitable cells only can be analyzed by Ca2+ imaging technique. Combined with recording of local field potential, we can simultaneously monitor local microcirculation, astrocytic Ca2+ signaling, and synaptic activity within the same field. We have observed that astrocytes, as opposed to neurons, are activated and display spontaneous Ca2+ oscillations, as well as propagating Ca2+ waves in the ischemic penumbra. Astrocytic Ca2+ signaling is associated with the release of several neurotransmitters, including glutamate and ATP.-ATP is, in the extracellular space, rapidly degraded to adenosine - anendogenous neuroprotective agent. On the basis of these observations, this proposal will test the proposition that astrocytic Ca2+ signaled- release of ATP, and the latter's rapid degradation to adenosine, mediates the electrical silence of the ischemic penumbra. In Aim 1, we will characterize ATP release in the setting of focal ischemia. Aim 2 intends to define astrocytic Ca2+ signaling in the penumbra, using 2-photon laser scanning microscopy to establish which transmitters (glutamate, ATP) trigger the abnormal Ca2+ signaling by local application of receptor antagonists. Aim 3 will directly image NADH to assess the cellular metabolic responses to reduced blood flow in the ischemic penumbra, and will define the respective contributions of astrocytes and neurons in this regard. Combined imaging of capillary flow and Ca2+ or NADH imaging will allow a correlation between local perfusion and astrocytic Ca2+ signaling or NADH on a single cell level in live animals. For these experiments, we will use of transgenic Thy1-YFP loaded with the astrocyte-specific indicator, sulforhodamine 101 mice. Aim 4 then asks if astrocytic Ca2+ signaling by release of ATP/adenosine reduce synaptic transmission, lower metabolic demands, and thereby increase neuronal survival in the penumbra. In specific, we will define the role of adenosine A1 receptors in synaptic depression in the penumbra. Preliminary observations show that the adenosine A1 receptor antagonist, DPCPX, triggered a robust increase in synaptic activity in the penumbra without a concomitant increase in blood flow. Our expectation is that a more precise mechanistic understanding of the role of astrocytes in this process will ultimately justify the development and assessment of adenosine mimetics and modulators as therapeutic agents in ischemic stroke. PHS 398 (Rev. 09/04) Page 71. Form Page 2 PO1 NS050315 Project 1 Principal Investigator/Program Director (Last, First, Middle): Nedergaard, Maiken Introduction to revised application This is a second revision of our PPG application entitled The role of astrocytes in ischemic stroke, and of my section therein, Astrocytic calcium signaling in the ischemic penumbra. The past submission of this proposal was praised for being highly innovative, and for addressing several fundamentally new areas in stroke research. However, the referees raised concerns regarding: 1) our technical ability to causally link acute ischemic events, e.g. ATP release and Ca2+ signaling, to neuronal death; 2) the concept of ATP as an excitatory transmitter, and its potential to trigger neuronal death in ischemia; and, 3) the specificity of P2X receptor antagonists. Based on the reviewers' comments, we have chosen to focus this revised application on acute cellular responses to ischemia, thereby taking maximal advantage of our 2-photon imaging approaches to assessing both intracellular and intercellular signaling events in situ. I have also dropped Aim 3, which correlated ATP release with delayed neuronal injury, and which was viewed as too preliminary by the referees. We have since last application refined the technique of in vivo NADH imaging in the ischemic penumbra. NADH is the principal electron carrier in glycolytic and oxidative metabolism, and is an intrinsic indicator of cellular redox state. Recent developments in 2-photon imaging have improved its spatial resolution substantially, so much so as to resolve subcellular changes in NADH within the ischemic cortex. Another advantage of 2-photon NADH imaging is that we can identify and isolate changes in redox state, in both individual cells and their processes. Use of Thy1-YFP mice, that selectively express YFP in central neurons, combined with loading of the astrocyte specific indicator, sulforhodamine 101, has enabled us to separately assess the respective metabolic responses of neurons and astrocytes within the same field of view. Our preliminary observations have indicated a strong compartmentalization of metabolic responses in the ischemic penumbra. Combined imaging of NADH and capillary flow should therefore enable us to precisely define changes in neuronal and astrocytic redox state, in response to experimentally-defined reductions in blood flow. By imaging NADH and Ca2+, we intend to assess the interdependence of hypoxia-associated NADH and Ca2+ increases. Our collaborator, Frank Kirchhoff, Gottingen, has established a set of spectrally distinct GFAP reporter mice that include GFAP-EGFP, GFAP-AMCyan, and GFAP-mRFP1, which will permit us to directly visualize astrocytes concurrently with NADH and Ca2+ imaging. In light of the upgrade of our 2-photon imaging setup to 3 channel detection, these mice will greatly facilitate the combined analysis of Ca2+, capillary perfusion, and NADH in astrocytes and neurons. By this means, we intend to assess the interaction between Ca2+ signaling and NADH levels among defined cell types within the ischemic penumbra. In our revised aim 3, 1intend to test the hypothesis that astrocytic ATP release comprises a conserved mechanism of cell protection. Our studies so far have suggested that adenosine's neuroprotective effects outweigh P2XR-mediated excitotoxic injury in the cortex. We found that P2X receptor antagonists have little effect upon neuronal injury in the penumbra, whereas adenosine receptor antagonists potently increased ischemic injury after MCA occlusion. ATP is released by astrocytes in response to reduced perfusion in the ischemic penumbra. Besides its activation of local purinergic receptors, it is also rapidly converted to adenosine, which potently inhibits excitatory transmission while lowering cellular energy demands. One of the defining characteristics of the ischemic penumbra is its electrical silence. On that basis, we suspect that neuronal activity is depressed by adenosine; as a corollary to this postulate, we have found that adenosine receptor antagonists increase electrical activity in the ischemic penumbra (Fig. 15). We believe these additions and modifications to our proposal allow us to take better advantage of our available imaging and molecular resources, while expanding both the breadth and rigor of our analysis of the role of astrocytes in stroke. Specific points: Reviewer 2 was unconvinced by the rationale for correlating rates of glycolysis with ATPrelease. We have removed this aim. The validity of the proposed experiments with BAPTA-AM and uncaging was questioned by reviewer2. Chelation of cytosolic Ca2+ with BAPTA is widely used approach to study the functional significance of Ca2+ signaling. BAPTA does efficiently block cytosolic Ca2+ increases, and has low toxicity. We agree with the reviewer that BAPTA-AM and uncaging are unphysiological approaches, but also would like to point out that they are excellent tools to define the impact of Ca2+ signaling. Photolysis of caged Ca2+ is considered state-of- the-art in study of astrocytes and we used this approach in a recent publication (Takano et al., 2006). Few approaches exist that selectively target astrocytes. Use of BAPTA and caged Ca2+ can add important PHS 398/2590 (Rev. 09/04) Page 72~ Continuation Format Page